Project Summary/Abstract In the previous project period we completed our 3D histomorphometric characterization of connective tissue deformation and remodeling in early through end-stage monkey experimental glaucoma (EG). Based on those findings, in this renewal proposal we will seek to identify important molecular and cellular components of optic nerve head (ONH) connective tissue and retrolaminar myelin remodeling in monkey early experimental glaucoma (EG). In so doing we will identify age-related differences in these phenomena and provide unprecedented microscopic support for their early in vivo detection by optical coherence tomography (OCT). In Aim 1 we will phenotype the ONHs of young and old monkeys with unilateral early EG using quantitative proteomic, immunohistochemistry (IHC), in situ hybridization (ISH) and electron microscopy (EM) techniques. We hypothesize that connective tissue and retrolaminar myelin remodeling are core components of ONH aging and glaucoma and will test predictions regarding their timing, and character in monkey early EG relative to the development of retinal ganglion cell (RGC) cytoarchitecture disruption, alterations in the ONH constituent cells, and the presence of wound healing, inflammation and monocyte/macrophage infiltration. In Aim 2 we will explore our hypothesis that the pathophysiology of ONH aging overlaps with glaucoma by testing two predictions regarding old vs young Control eye differences in the Aim 1 data: first that they will share important features of EG vs Control eye differences in monkeys of all ages; and second that they will also share important features of old vs young EG eye differences in monkey early EG. In Aim 3 we will accurately colocalize Aim 1 IHC/ISH/EM findings to sacrifice-day OCT B-scans of the same eyes so as to provide light and electron microscopic validation in monkeys for ONH OCT detection of early glaucoma damage in humans. To accomplish these aims we will employ: state of the art quantitative iTRAQ Mass Spectroscopy; our novel strategy for quantitative ONH IHC/ISH; quantitative transmission and scanning block face EM; precise anatomic colocalization of each IHC/ISH paraffin and each EM vibratome section to OCT B-scans, retrolaminar optic nerve (ON) axon counts, and sacrifice day OCT change-from-baseline data. All outcomes will be reported in anatomically consistent, 30 degree ONH sectors that are oriented relative to the foveal-BMO axis. Aim 1 will generate the first quantitative proteomic characterization of the monkey ONH, peripapillary scleral (pp-scleral), ON and retinal tissues in early EG. IHC/ISH and EM studies will demonstrate that connective tissue and myelin remodeling are present at an early stage of RGC axonal insult. They will also detect alterations in the ONH and pp-scleral tissues and cells that include proliferation, phagocytosis, myofibroblast differentiation, wound healing, monocyte infiltration, and inflammation. Aim 2 will confirm that proteomic, IHC protein expression and EM differences in old versus young control eyes share important features with early EG vs control eye differences in monkeys of all ages. Aim 3 will provide the first rigorous microscopic validation of deep ONH visualization and early glaucoma ONH change detection by OCT.